Physiological and Transcriptional Characteristics of Banana Seedlings in Response to Nitrogen Deficiency Stress

Round 1

Reviewer 1 Report

General comments :

The authors reported the change in gene expression and physiology of banana seedlings under the treatment of nitrogen deficiency.  Overall, the manuscript contains interesting information and it is original. However, I have several comments and suggestions below.

Specific comments :

Line104. How to calculate the nitrogen concentration for treatment? What kind of substance added and its form ? Please elaborate.

Line 106. No supplementary Table S1

Line 183. Figure 1A. Please include the information of bar scale.

Line 188. Please include the information regarding the post hoc test for one-way ANOVA analysis.

Line 234. Please include the information regarding the post hoc test for one-way ANOVA analysis.

Line 255. No supplementary Figure S1

Line 268. No supplementary Figure S2

Line 270. Figure 4B, please include Y-axis label. For instance, no of genes.

Line 476. There is no supplementary. Table S1, S2 and S3 submitted with the manuscript . Please include these documents in the revised manuscript.  

Tables S2 and S3 not mentioned in the main manuscript.

Figure 5B, 6B,7B, 8B. please include Y-axis label.

Figure 5B, 6B,7B, 8B. The authors need to compare the qRT-PCR results with RNA-seq (FPKM) for validation, whether they have similar results or no?. Please include this  information in the Figure B. For instance, left Y-axis fold change and right Y-axis FPKM.

Line 463. The conclusion is not really based on data presented. Please revise this sentence “Low N stress induced various plant hormone signals, including GA, SL, IAA, and ABA”. Since the authors do not measure these hormones directly but only its expression level. 

Minor editing of English language required

Author Response

Thank you very much for your valuable comments. According to your comments, we have revised the shortcomings in the manuscript one by one.

1. Comment: Line104. How to calculate the nitrogen concentration for treatment? What kind of substance added and its form? Please elaborate.

Response: I am very sorry that the attached table S1 was missed before. In fact, we have detailed the composition and concentration of the added substance in Schedule S1 and re-uploaded it.

2. Comment: L:106, 255 et supplementary materials。

Response: We have revised and uploaded the supplementary file..

3. Comment: Line 183. Figure 1A. Please include the information of bar scale.

Response: We have filled in the missing information in Figure 1A.

4. Comment: Line 188, 234. Please include the information regarding the post hoc test for one-way ANOVA analysis.

Response: According to your suggestion, we have added the relevant information.

5. Comment: Line 270. Figure 4B, please include Y-axis label. For instance, no of genes.

Response: We have redrawn Figure 4B and added the Y-axis label.

6. Comment: Figure 5B, 6B,7B, 8B. The authors need to compare the qRT-PCR results with RNA-seq (FPKM) for validation, whether they have similar results or no?. Please include this information in the Figure B. For instance, left Y-axis fold change and right Y-axis FPKM.

Response: We have reanalyzed and added the FPKM results in Figure 5B, 6B, 7B, and 8B. Meanwhile, the relevant descriptions have been added in the text.

7. Comment: Line 463. The conclusion is not really based on data presented. Please revise this sentence “Low N stress induced various plant hormone signals, including GA, SL, IAA, and ABA”. Since the authors do not measure these hormones directly but only its expression level.

Response: According to your suggestion, we have revised the relevant description. “Low nitrogen stress induced the expression of many plant hormone-related genes such as GA, SL, IAA and ABA”.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript concerns the effects of various nitrogen levels on the growth, photosynthetic characteristics and gene transcription in banana seedlings. The choice of banana as an experimental model for this investigation has been justified by its economical importance. The Authors claim that the results can be treated as theoretical basis for trials of fertilization in banana production (however, the experiments were performed in in vitro conditions).

The manuscript is generally well written and well presented. The methods has been selected properly, the investigation was well designed. The results are clearly presented (an exception is described below), the discussion is convincing.

Required corrections:

The methods are generally well described, however, the meaning of SPAD (chapter 3.2) and the method of its determination (chapter 2.2) is not clearly presented. The abbreviation SPAD is explained only in the Abstract (please repeat it in Material and Methods), but the real meaning of “Specialty Products Agricultural Division” (what is means, why and how it is determined?) is not obvious for all the Readers. It requires better explanation.

Please also correct the Figure 9 (Phtotsynthetic) ? If it is supposed to mean a physiological mechanism, it should be either photosynthesis or photosynthetic capacity?

Author Response

Thank you very much for your valuable comments. According to your comments, we have revised the shortcomings in the manuscript one by one.

1. Comment: The methods are generally well described, however, the meaning of SPAD (chapter 3.2) and the method of its determination (chapter 2.2) is not clearly presented. The abbreviation SPAD is explained only in the Abstract (please repeat it in Material and Methods), but the real meaning of “Specialty Products Agricultural Division” (what is means, why and how it is determined?) is not obvious for all the Readers. It requires better explanation.

Response: According to your suggestions, we have added or modified the relevant description of SPAD in this text.

2. Comment: Please also correct the Figure 9 (Phtotsynthetic) ? If it is supposed to mean a physiological mechanism, it should be either photosynthesis or photosynthetic capacity?

Response: Thank you for pointing out this error, we have corrected it.

Author Response File: Author Response.pdf

Reviewer 3 Report

Dear Authors,

The focus of the manuscript entitle “Physiological and transcriptional characteristics of banana  seedlings in response to nitrogen-deficiency stress“ is to elucidate the physiological and molecular mechanism under nitrogen-deficiency stress in Musa acuminata - banana plant. The research was conducted into the effects of various N levels on the growth, photosynthetic characteristics and gene transcription levels in banana seedlings.

The manuscript is relevant for the field. It is presented in a well-structured manner. The cited references are relevant and mostly recent publihsed. The manuscript is with scientific relevance and the experimental design is appropriate.

However, I find some flaws in the manuscript, that need to be removed/imroved to be suitable for publicatio:

·        The manuscript’s results could be reproducible if the Authors give some more details in the methods section. M&M need to we improved with more details.

·        In M&M 2.1 - It is not clear what kind of samples did you collect and for what? For how long? You have written “every week”, is that mean every week for the 30 days while the plants were rooted or not?

·        In M&M 2.7 – You need to mention which genes you analysed for expression pattern. How you chose the genes for the expression analyses? How you design the primers for PCR analysis? Where are given the primers? This information is missing.

·        In Results, try to stick to a description of the results you obtained without discussing them or drawing conclusions. Leave the reasoning and comparisons for the discussion.

·        Figures S1 and S2 are missing

·        The data from Figure 3 (E) and Figure 4 (C, D, E) it is not interpreted appropriately and consistently throughout the manuscript.

·        Figure 5 (B) is not interpreted appropriately and consistently throughout the manuscript. The results from Fig 5 (B) are not explain in the text. How you decide to analyse these genes for expression pattern? Also, the legend for Fig. 5(A) should be placed after Fig. 5(A) for clarity.

·        The same remark applies to Fig. 6, 7 and 8.

·        The Results from qRT-PCR are not explained or commented on at all, neither in the Results nor in the Discussion. This is a big omission. Please add this information to the manuscript or remove the data from the RT-PCR analysis altogether.

·        Depending on this, the conclusions should also be changed and improved.

English language is fine. There are some minimum grammar and punctuation errors in the text

I recommend major revision.

Author Response

Thank you very much for your valuable comments. According to your comments, we have revised the shortcomings in the manuscript one by one.

1. Comment: In M&M 2.1 - It is not clear what kind of samples did you collect and for what? For how long? You have written “every week”, is that mean every week for the 30 days while the plants were rooted or not?

Response: The main banana variety (Musa acuminata) is mainly propagated by asexual reproduction. In our experiment, tissue culture seedlings with three leaves and roots were used for sand culture. Each plant was added with 300 mL nutrient solution of different nitrogen concentrations per week (Table S1). After 28 d, root samples collected were immediately frozen in liquid nitrogen and were stored at -80℃.

We are sorry for the error in our description here, which has been modified in the text.

2. Comment: In M&M 2.7 – You need to mention which genes you analysed for expression pattern. How you chose the genes for the expression analyses? How you design the primers for PCR analysis? Where are given the primers? This information is missing.

Response: We have supplemented the information on selected genes and primers in Supplementary Table S2. We are very sorry that we did not upload the attachment at first.

In addition, we have added related descriptions in the Materials Methods section.

3. Comment: In Results, try to stick to a description of the results you obtained without discussing them or drawing conclusions. Leave the reasoning and comparisons for the discussion. Response: That's good advice. We have removed some of the discussion in the results section as much as possible. But some paragraph concluding sentences have been retained. Hope to get your approval.
4. Comment: Figures S1 and S2 are missing.

Response: I'm sorry that we missed the attached materials, now we have added and uploaded.

5. Comment: The data from Figure 3 (E) and Figure 4 (C, D, E) it is not interpreted appropriately and consistently throughout the manuscript.

Response: According to your suggestion, we have added the descriptions of these figures.

6. Comment: Figure 5 (B) is not interpreted appropriately and consistently throughout the manuscript. The results from Fig 5 (B) are not explain in the text. How you decide to analyse these genes for expression pattern? Also, the legend for Fig. 5(A) should be placed after Fig. 5(A) for clarity. The same remark applies to Fig. 6, 7 and 8.

Response: According to your suggestion, we have supplemented the relevant analysis and description in Figures 5, 6, 7, and 8. Meanwhile, we have also modified the position of the legend in these figures.

7. Comment: The Results from qRT-PCR are not explained or commented on at all, neither in the Results nor in the Discussion. This is a big omission. Please add this information to the manuscript or remove the data from the RT-PCR analysis altogether.

Response: Thank you for your very good suggestion. Indeed, we are very sorry that the results of the qRT-PCR analysis were missed. We have reanalyzed the qRT-PCR data and added detailed descriptions in 3.5, 3.6, 3.7, and 3.8 results sections. Meanwhile, the relevant analysis and description have been supplemented in the discussion section.

8. Comment: Depending on this, the conclusions should also be changed and improved.

Response: According to your suggestion, we have revised the relevant descriptions in discussion section.

9. Comment: English language is fine. There are some minimum grammar and punctuation errors in the text.

Response: We have carefully checked and revised all the descriptions and punctuation in the whole text.

Round 2

Reviewer 3 Report

Dear Authors,

I am satisfied with the changes and corrections made to the text. The missing information, figures and tables are added. In my opinion, the manuscript is ready for publication in its current form.